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Haptoglobin, alpha-thalassaemia and glucose-6-phosphate 
dehydrogenase polymorphisms and risk of abnormal 
transcranial Doppler among patients with sickle cell 
anaemia in Tanzania 



Sharon E. Cox, 1 ' 2 Julie Makani, 2 ' 3 
Deogratias Soka, 2 Veline S. 
L'Esperence, 4 Edward Kija, 2 Paula 
Dominguez-Salas, 1 Charles R. J. 
Newton, 2 ' 5 ' 6 Anthony A. Birch, 7 Andrew 
M. Prentice 1 and Fenella J. Kirkham 8 
*MRC International Nutrition Group, London 
School of Hygiene & Tropical Medicine, London, 
UK, 2 Muhimbili Wellcome Programme, Muhim- 
bili University of Health & Allied Sciences, Dar 
es Salaam, Tanzania, 3 Nuffield Department of 
Clinical Medicine, University of Oxford, Oxford, 
4 Faculty of Medicine, University of Southampton, 
Southampton, 5 Department of Psychiatry, Uni- 
versity of Oxford, Oxford, UK, 6 Kenya Medical 
Research Institute (KEMRI) Centre for Geo- 
graphic Medicine Research (Coast), Kilifi, Kenya, 
7 Neurological Physics Group, Department of 
Medical Physics and Bioengineering, University 
Hospital Southampton NHS Foundation Trust, 
Southampton, and s Institute of Child Health, 
University College London, London, UK 

Received 24 October 2013; accepted for 
publication 5 January 2014 
Correspondence: Dr Sharon Cox, Muhimbili 
Wellcome Programme, Muhimbili University 
of Health and Allied Sciences, PO Box 65001, 
Dar-es-Salaam, Tanzania. 
E-mail: Sharon.cox@muhimbili-wellcome.org 



Summary 

Transcranial Doppler ultrasonography measures cerebral blood flow veloc- 
ity (CBFv) of basal intracranial vessels and is used clinically to detect stroke 
risk in children with sickle cell anaemia (SCA). Co-inheritance in SCA of 
alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) poly- 
morphisms is reported to associate with high CBFv and/or risk of stroke. 
The effect of a common functional polymorphism of haptoglobin (HP) is 
unknown. We investigated the effect of co-inheritance of these polymor- 
phisms on CBFv in 601 stroke-free Tanzanian SCA patients aged <24 years. 
Homozygosity for alpha-thalassaemia 3-7 deletion was significantly associ- 
ated with reduced mean CBFv compared to wild-type (p-coefficient 
— 16-1 cm/s, P = 0 002) adjusted for age and survey year. Inheritance of 1 
or 2 alpha-thalassaemia deletions was associated with decreased risk of 
abnormally high CBFv, compared to published data from Kenyan healthy 
control children (Relative risk ratio [RRR] = 0-53 [95% confidence interval 
(CI):0-35-0-8] & RRR = 0-43 [95% CI:0-23-0-78]), and reduced risk of 
abnormally low CBFv for 1 deletion only (RRR = 0-38 [95% CI:0T7- 
0-83]). No effects were observed for G6PD or HP polymorphisms. This is 
the first report of the effects of co-inheritance of common polymorphisms, 
including the HP polymorphism, on CBFv in SCA patients resident in 
Africa and confirms the importance of alpha-thalassaemia in reducing risk 
of abnormal CBFv. 

Keywords: sickle cell disease, Africa, children, cerebral blood flow velocity. 



Although sickle cell anaemia (SCA) is a monogenic disorder 
caused by the homozygous inheritance of sickle haemoglobin 
(HbS) resulting in haemolytic anaemia, there is a wide varia- 
tion in the severity and pattern of morbidities (Beutler, 
2001). The contributions of other co-inherited genetic vari- 
ants on risk of severe outcomes such as stroke are under 
investigation (Sebastiani et al, 2005; Flanagan et al, 2011). 
Stroke is reported to occur in 11% of patients aged <20 years 
(Ohene-Frempong et al, 1998; Pandey & Gorelick, 2005). 



Transcranial Doppler ultrasonography (TCD) is a well-estab- 
lished measure of cerebral blood flow velocity (CBFv) of 
basal intracranial vessels and abnormal CBFv may be second- 
ary to increased cerebral blood flow or to decrease in diame- 
ter of the vessel secondary to vasospasm or stenosis. The 
technique is used clinically to detect SCA patients with a 
high risk of stroke (Adams et al, 1992) with velocities of 
>200 cm/s and >170 cm/s predicting 40% and 7% risk of 
stroke over the subsequent 3 years without treatment 



© 2014 The Authors. British Journal of Haematology Published by John Wiley & Sons Ltd. First published online 21 February 2014 

British Journal of Haematology, 2014, 165, 699-706 doi:1 0.1 1 1 1/bjh. 12791 

This is an open access article under the terms of the Creative Commons Attribution License, which permits 
use, distribution and reproduction in any medium, provided the original work is properly cited. 



BRITISH JOURNAL 
OF HAEMATOLOGY 




S. E. Cox ef al 



(Adams et al, 1992). The risk is substantially reduced by 
chronic blood transfusion (Adams et al, 1998). Elevated 
CBFv is also associated with increased risk of other neurolog- 
ical complications, including seizures (Prengler et al, 2005), 
neurocognitive deficits (Krai et al, 2003; Strouse et al, 2006) 
and silent infarcts (Pegelow et al, 2001). Low velocities may 
also be seen in patients with cerebrovascular disease, consis- 
tent with proximal vascular stenosis (Kirkham et al, 1986) or 
occlusion (Lee et al, 2004) and appears to be associated with 
cerebrovascular accidents (Buchanan et al, 2012). 

Haptoglobin (HP) is an acute phase protein that removes 
free haemoglobin (Hb) from the circulation after haemolysis 
or tissue damage, and is therefore an important anti-oxidant. 
HP is encoded by the co-dominant alleles, HP*1 and HP*2, 
resulting in three distinct phenotypes HP11, HP12 and 
HP22. Plasma concentrations of HP, and its binding affinities 
for free Hb, vary by phenotype, with HP22 reported to have 
reduced overall Hb affinity (Langlois & Delanghe, 1996), less 
efficient antioxidative capacity, (Langlois & Delanghe, 1996; 
Melamed-Frank et al, 2001) and reduced clearance rates of 
HP2-Hb complexes by macrophages (Asleh et al, 2003). This 
is suggested to result in increased nitric oxide scavenging by 
HP2-Hb complexes (Azarov et al, 2008), with reduced down- 
stream anti-inflammatory signalling after macrophage endo- 
cytosis of HP2-Hb complexes (Landis et al, 2013), weaker 
inhibition of prostaglandin synthesis and a stronger angio- 
genic effect (Cid et al, 1993). There is strong evidence that 
the HP22 phenotype is associated with increased risk of 
stroke and cardiovascular events in diabetic patients (Vardi 
et al, 2012). Furthermore, several reports demonstrate an 
association between the HP*2allele with vasospasm after 
subarachnoid haemorrhage; as determined by high CBFv 
(Borsody et al, 2006), angiographically (Ohnishi et al, 2013), 
and in a mouse model (Chaichana et al, 2007). Vasospasm 
after sub-arachnoid haemorrhage is thought to result from 
inflammation, oxidative stress, nitric oxide scavenging, leuco- 
cyte migration and endothelial-leucocyte interactions (Chai- 
chana et al, 2010), all from extracellular Hb. In HP22 mice 
with sub-arachnoid haemorrhage, treatment with an anti-oxi- 
dant (Froehler et al, 2010) or controlled nitric oxide (Momin 
et al, 2009) reduces vasospasm, which is supportive evidence 
that these mechanisms are important. The HP*2allele is also 
associated with increased severity and adverse outcomes in 
various infections including human immunodeficiency virus 
(HIV and malaria (Cox et al, 2007; McDermid & Prentice, 
2006). Effects of HP phenotype on cerebrovascular outcomes 
in non-diabetic patients, e.g. those with SCA, have not been 
reported. 

Although the co-inheritance of oc-thalassaemia deletions 
and glucose-6-phosphate dehydrogenase (G6PD) deficiency 
have been previously investigated as disease modifiers in 
SCA, specifically affecting risk of elevated CBFv and/or 
stroke, these have not been investigated in Africa. The 
co-inheritance of alpha-thalassaemia in SCA modifies red cell 
indices (Embury et al, 1984; Stevens et al, 1986; Kulozik 



et al, 1988) and red cell rheology (Serjeant et al, 1983), and, 
in some reports, increases total Hb (Embury et al, 1982). 
Alpha-thalassaemia is consistently associated with a decreased 
risk of increased CBFv and/or stroke in SCA (Adams et al, 
1994; Hsu et al, 2003; Bernaudin et al, 2008; Belisario et al, 
2010; Flanagan et al, 2011). G6PD deficiency results in 
decreased capacity to reduce oxidized glutathione via NADPH 
and thus reduced ability of red cells to counteract oxidant 
stress (Mason et al, 2007). G6PD deficiency (A-genotype) in 
SCA is associated with lower Hb but not increased haemolysis 
(Nouraie et al, 2010). Reports of the effects of co- inheritance 
of G6PD deficiency with CBFv, vasculopathy on magnetic 
resonance angiography and/or stroke risk are contradictory 
(Bernaudin et al, 2008, 2011; Rees et al, 2009; Miller et al, 
2011; Thangarajh et al, 2012) and there are no data from 
patients resident in Africa. 

We hypothesized that the co-inheritance of the HP*2allele 
in children with SCA would increase the risk of high CBFv 
and furthermore, that there may be epistatic effects between 
the three unrelated polymorphisms under investigation such 
that an effect may be greatest in, or limited to, children with 
the HP*2allele who also co-inherited G6PD deficiency and 
did not have alpha thalassaemia. 

Methods 

Ethical approval was granted by the Muhimbili University of 
Health and Allied Sciences, Tanzania (MU/RP/AEC/VOL XI/ 
33), and the London School of Hygiene and Tropical Medi- 
cine, UK (reference 5158) Review Boards. All participants, 
parents or guardians gave written informed consent (in 
Kiswahili) for participation at enrolment into the SCA clini- 
cal cohort at Muhimbili National Hospital (MNH). 

Study population and Transcranial Doppler examination 

Participants aged <24 years were enrolled in the Muhimbili 
Sickle Cohort at Muhimbili National Hospital, Tanzania 
and had a TCD examination at one or more of three cross- 
sectional surveys, conducted in 2004/05, 2009 and 2010. 
Patients who received blood transfusion within the previous 
two months, symptoms of sickle crisis within the previous 
2 weeks or a previous history of stroke were excluded. TCD 
examinations were performed according to the STOP 
(Stroke prevention in sickle cell disease) protocol (Nichols 
et al, 2001) using the Companion II (Nicolet, Warwick, 
UK). CBFv was determined as the highest time averaged 
maximum velocity in the distal internal carotid artery and 
middle cerebral artery on either side. CBFv was classified 
using data from 115 Kenyan non-SCA control children 
(Newton et al, 1996) as low (<43 or <39-5 cm/s) for the 
left and right middle cerebral artery (LMCA/RMCA), high 
(>141 or >143-5 cm/s LMCA/RMCA) or normal. CBFv 
were also classified as conditional (170-199 cm/s) or abnor- 
mal (>200 cm/s) based on previous criteria (Adams et al, 



700 



© 2014 The Authors. British Journal of Haematology Published by John Wiley & Sons Ltd. 

British Journal of Haematology, 2014, 165, 699-706 



Polymorphisms & Transcranial Doppler in SCA 



1990; Newton et al, 1996). In addition, the difference 
between the maximum velocities in the ipsilateral to contra- 
lateral MCA was calculated; this was previously shown to 
predict vasospasm in patients after sub-arachnoid haemor- 
rhage (Nakae et al, 2011). The difference between the two 
sides was then calculated as a percentage of the lower 
velocity and categorized to indicate the degree of asymme- 
try between the left and right MCA as: no asymmetry - 
values less than 40%, degree 1; values 40-75%, degree 2; 
75-180%, degree 3; and, >180%, degree 4. These cut-off 
points are currently used clinically to follow vasospasm 
after subarachnoid haemorrhage at University Hospital 
Southampton. 

Laboratory procedures 

Sickle status was diagnosed and quantification of Hb frac- 
tions was performed by high performance liquid chromatog- 
raphy (HPLC) using the /?-thalassaemia Short Program on 
the Variant I analyser (BioRad, Hercules, CA, USA). Full 
blood counts were performed using an automated cell coun- 
ter (Pentra 60, Horiba ABX, Kyoto, Japan) within 7 d of the 
TCD study. Genomic DNA was isolated from peripheral 
blood leucocytes using Nucleon kits (BACCII). HP functional 
variants (alleles HP*1 and HP*2) were genotyped by allele- 
specific polymerase chain reaction (PCR) adapted from 
published techniques (Koch et al, 2002; Cox et al, 2007). 
Individuals were genotyped for the 3-7 alpha-thalassaemia 
deletion using a PCR-based method and agarose gel visuali- 
zation as per published methods (Williams et al, 2005). The 
202- and 376-single nucleotide polymorphisms (SNPs) 
(rsl050828 [G-202A] & rsl050829 [A-376G]), the combined 
inheritance of which results in the A- phenotype of glucose 
6-phosphate dehydrogenase (G6PD) deficiency, and HbS 
(rs334) were determined using multiplex Sequenom® Mass- 
ARRAY® (Sequenom®, Hamburg, Germany). 

Statistical analysis and sample size 

In the current analysis one observation per subject was 
selected according to the observation that recorded the high- 
est CBFv, assessed separately in the l- and RMCA. Statistical 
analyses were performed with Stata IC software (version 
12.0, StataCorp, College Station, TX, USA). Categorical vari- 
ables were constructed for the CBFv values, using the Kenyan 
and STOP cut-off points as described above. We used two 
approaches to analyse the CBFv outcome. In the first we 
used multiple linear regression to explore predictors of CBFv 
as a continuous variable, whilst excluding those with absent 
signal. We determined that, using this approach, 90 children 
per group (HP22 vs. HP11) were required to detect a differ- 
ence in mean CBFv equivalent to 0-5 x standard deviation 
(SD) of mean CBFv, based on data from similar aged Kenyan 
children with SCA (Makani et al, 2009) with 90% power and 
two-tailed significance at 5%. In the second approach, we 



used multinomial logistic regression to explore predictors of 
CBFv as a categorical variable representing potential cardio- 
vascular disease (CVD) (allowing simultaneous investigation 
of factors affecting risk of low vs. normal and high vs. low 
CBFv, presenting the results as relative risk ratios (RRRs) 
with 95% confidence interval (CI). A P-value <005 was 
considered statistically significant. 

Results 

Characteristics of the study population 

A total of 601 homozygous SCA patients were included. The 
demographic, laboratory and CBFv characteristics of the 
patients are summarized in Table I. The age of the patients 
ranged from 0-6 to 22-6 years, with 12% aged <5 years and 
2-5% aged >16 years. The majority of subjects (51%) were 
included from the largest survey in 2004/2005. There was no 
evidence of a difference between the surveys for sex, geno- 
type or Hb concentrations. However, mean age and HbF% 
were significantly lower in the 2004/05 survey. All the partici- 
pants had genotype results for at least one of the three poly- 
morphisms under investigation, with 385 having complete 
data for the three genes being investigated. 

Mean CBFv was 131 cm/s (SD = 42). The proportion of 
non-detectable CBFv in the LMCA compared to the RMCA 
was significantly different (P < 0-006) at 5% for the LMCA 
and 8% for the RMCA. However, there was no significant 
difference in mean CBFv between the left and right MCA 
(120 cm/s [SD 42, N = 571] vs. 119 cm/s [SD 42, N = 552]). 

The genotype prevalence data are summarized in Table II. 
The prevalence of the inherited combinations of the three 
genes in the 385 participants with a complete dataset is avail- 
able as supplementary material in Table SI. 

Factors associated with TCD outcomes 

In the first approach we used linear regression to investigate 
possible associations with CBFv for age, sex, Hb and HbF%, 
as well as the three genes under investigation, including all 
measureable CBFv observations. The results are summarized 
in Table III. Strong inverse associations with CBFv were 
apparent for age and Hb. Initial analyses of the genotypes 
were adjusted for age and year of survey. Hb concentration 
was not immediately adjusted for, as all of the genotypes 
under investigation may potentially act, at least in part, 
through effects on Hb. Inheritance of two copies of the 3-7 
cx-thalassaemia deletion, compared to no copies, was signifi- 
cantly associated with decreased mean CBFv. If Hb was 
adjusted for, no effect of ot-thalassaemia was observed, sug- 
gesting the effect is mediated via Hb. There were no apparent 
effects of G6PD status or HP genotype, even when when 
limited to those with gene combinations hypothesized as the 
'worst' and 'best combinations ([HP22/A-A-or A-Z, and/or, 
aa/aa] vs. [HPll/BB/-a/-a]), 



© 2014 The Authors. British Journal of Haematology Published by John Wiley & Sons Ltd. 
British Journal of Haematology, 2014, 165, 699-706 



701 



S. E. Cox ef al 



Table I. Patient characteristics. 





Summary 


Observations 


Demographic variables 


statistic 


(N) 


Age [years], mean (SD) 


9-76 (3-86) 


601 


Males, n (%) 


325 (54-08) 


601 


Survey, n (%) 






2004/05 


305 (50-75) 


601 


2009 


224 (37-27) 




2010 


72 (11-98) 




Laboratory variables* 






Haemoglobin [g/1], mean (SD) 


74-0 (11-2) 


583 


Fetal haemoglobin [%], mean (SD) 


5-23 (3-84) 


533 


CBFv 






MCA maximum velocity, left or right, 


n (%) 




Kenyan non-SCA criteriaf 




601 


Normal 


387 (64-39) 




Low [<43 cm/s or <39-5 cm/s] 


44 (7-32) 




High [>141 cm/s or >143-5 cm/s] 


170 (28-29) 




STOP criteria 






Normal 


534 (88-85) 




vjOnanionai ^i/u v zuu cm/s 


1^ (A 1 
ZD ( I - 10) 




Abnormal >200 cm/s 


42 (6-99) 




Asymmetry (Vasospasm):]: n (%) 






No asymmetry 


317 (54-75) 


579 


Mild asymmetry 


104 (17-96) 




Moderate asymmetry 


93 (16 06) 




Severe asymmetry 


65 (11-23) 





SD, standard deviation; CBFv, cerebral blood flow velocity; SCA, 
sickle cell anaemia; STOP, stroke prevention in sickle cell disease; 
MCA: middle cerebral artery. 

*A11 laboratory values were assessed within 7 d of the Transcranial 
Doppler (TCD) measurement, except HbF which if measured under 
the age of 60 months and not within 7 d of the TCD was not 
included. 

fBased on values from healthy Kenyan children (mean ± 2 SD) 
(Newton et al, 1996). 

^Degree of asymmetry is difference in the maximum velocities in the 
left MCA and right MCA, calculated as a percentage of the lower 
velocity. Mild = 40-75%, moderate - 75-180%, severe >180%. 

In the second approach we explored associations using mul- 
tinomial logistic regression, in which CBFv was categorized as 
low, normal or high (Table IV). The only genotype with evi- 
dence of an association was the 3-7 a-thalassaemia deletion, 
the inheritance of which was associated with a significantly 
decreased risk of having an abnormally high or low CBFv. 

We also investigated genotype associations with abnormal 
CBFv according to the STOP classifications (normal 



Table HI. Associations between demographic, laboratory variables 
and genetic variants with CBFv as a continuous measurement*. 

Observations 



Predictors (3 


-Coefficient (95% CI) 


P-value 


(n) 


^cr- \y J 1 


-2-28 (• 


-3-21 to -1-35) <0 0001 


525 


Qpv (malp vq 


3-512 (- 


-3-56-10-58) 


0-329 


525 


female) t 










Haemoglobin 


-4-60 ( 


-7-91 to-1-29) 


0007 


525 


(g/Dt 










Fetal 


0-197 ( 


-1-11-0-72) 


0-672 


525 


haemoglobin 










(%)t 










Haptoglobin HP lit 










HP12 


0-28 (■ 


-9-61-10-17) 


0-956 


410 


HP22 


■2-994 (■ 


-15-32-9-33) 


0-633 




ot-Thalassaemia normal^ 








1 Deletion 


-6-32 ( 


-13-78-1-14) 


0097 


549 


2 Deletions - 


1614 ( 


-26-271 


0002 






to -6 0135) 






G6PD Phenotype normal^ 








Mild (A-B) 


-8-80 (■ 


-19-44-1-84) 


0105 


564 


Affected (A-Z 


-1-81 ( 


-11-83-8-21) 


0-723 





or A-A-) 

*Only velocity values >0 cm/s included for multiple linear regression. 
fResults for multivariable regression including the marked variables. 
^Adjusted for age and survey year CBFv, cerebral blood flow velocity; 
95% CI, 95% confidence interval. 

<170 cm/s, conditional 10-199 cm/s and abnormal >200 cm/ 
s) (Adams et al, 1998) but no significant associations were 
observed. 

Finally we also investigated possible associations with the 
degree of asymmetry in CBFv, classified as normal, mild, 
moderate or severe. No consistent effects were observed for 
any of the variables tested (Table V). 

Discussion 

This is the largest study to date to assess the effects of the 
disease modifying and commonly co-inherited polymor- 
phisms of alpha-thalassaemia and G6PD on prospectively 
measured CBFv in SCA, and the first in children and adoles- 
cents resident in Africa. In addition, this is the first study to 
assess the effect of co-inheritance of HP polymorphisms on a 
clinical end-point in SCA. We confirm previous reports of a 
reduction in mean CBFv and protection from abnormal 
CBFv measurements in heterozygote and homozygotes for 



Table II. Genotype prevalence and scores assigned to each genotype. 



Gene 


Homozygote WT 


Heterozygote 


Homo/hemizygote mutant 


G6PD A-Phenotype (202 & 376) N = 583 


BB [75-30%] 


A-B [11-32%] 


A-Z or A-A-[13-38%] 


3-7 Alpha-thalassaemia deletion N = 568 


aalaa [43-66%] 


tx-/acx [40-67%] 


(X-/<X- [15-67%] 


HPN = 422 


HP11 [23-46%] 


HP12 [56-64%] 


HP22 [19-91%] 



702 



© 2014 The Authors. British Journal of Haematology Published by lohn Wiley & Sons Ltd. 

British Journal of Haematology, 2014, 165, 699-706 



Polymorphisms & Transcranial Doppler in SCA 



Table IV. Relative risk ratios of genetic variants on CBFv categories 
defined as low or high compared to normal using data from Kenyan 
healthy non-SCA child population adjusted for age and year of 
survey. 

Relative risk ratio 

(95% confidence interval)* 



Genotypes 



(CBFv Low 
vs. normal) 



(CBFv High 
vs. normal) 



iV 



Haptoglobin, HP 11 

HP12 0-90 (0-33-2-47) 

HP22 1 01 (0-30-3-38) 

oi-Thalassaemia, normal 

1 Deletion 0-38 (0-17-0-83)f 

2 Deletions 0-95 (0-41-2-20) 
G6PD Normal phenotype 

Mild 0-49 (0-14-1-65) 

Affected 0-41 (1-12-1-37) 



90 (0-53-1-51) 
85 (0-44-1-66) 

53 (0-35-0-80) + 
43 (0-23-0-78)f 



62 (0-32-1-17) 
78 (0-44-1-39) 



422 



568 



583 



*Low CBFv corresponds to values <43 in LMCA or <39-5 in RMCA, 
including values of 0; High CBFv corresponds to values >141-1 in 
LMCA or >143-6 in RMCA), compared to normal (CBFv: 43- 
141 cm/s in LMCA or 39-6-143-5 cm/s in RMCA) (Newton et al, 
1996). 

tP-value <0 05. 

RRR: Relative risk ratio; CBFv, cerebral blood flow velocity; SCA, 
sickle cell anaemia; RMCA, right middle cerebral artery; LMCA, left 
middle cerebral artery. 

the oe-thalassaemia 3-7 deletion. This effect was not signifi- 
cant when limited to abnormal, as defined by the STOP 
criteria. We also observed a significantly decreased risk of 
abnormally low CBFv in alpha thalassaemia heterozygote 
children. No effects of G6PD or HP polymorphisms were 



observed, including when assessed in children with the 
hypothesized 'worst' gene combinations compared to 'opti- 
mal' combination. 

Mean CBFv (131 cm/s) and the prevalence of highly 
elevated (>200 cm/s) (7%) and conditionally elevated (170- 
199 cm/s) (4%) CBFv were higher in our Tanzanian patients 
compared to rural Kenyan children with SCA with a mean 
CBFv of 120 cm/s and only 3% with conditional and none 
with high CBFv (Makani et al, 2009), but still considerably 
lower than in other studies in which alpha-thalassaemia and/ 
or G6PD were also assessed. Sixteen percent of French chil- 
dren with sickle cell disease had CBFv >200 cm/s (62/373) 
(Bernaudin et al, 2008), whilst 18% (409/2334) had CBFv of 
170-199 cm/s and 9% >200 cm/s (217/2324) in the baseline 
measurement of the STOP study in the USA (Adams et al, 
2004). However, the proportions were similar to that 
reported in Brazilian patients with SCA/sickle cell disease in 
whom 4% (7/164) had CBFv 170-199 cm/s and 3% (5/164) 
had a CBFv >200 m/s (Belisario et al, 2010). In both the 
Brazilian and the French cohorts the 3-7 alpha-thalassaemia 
deletion was associated with protection from conditional or 
high CBFv, whilst in the STOP trial cohort a similar finding 
was observed in a case control design (Hsu et al, 2003). Thus 
our results confirm a protective effect of the alpha-thalassae- 
mia 3-7 deletion, resulting in a lower mean CBFv and reduc- 
ing the risk of even moderate elevations, compared to 
normal, and importantly, also protecting against abnormally 
low CBFv, also an indicator of CVD and stroke risk. How- 
ever, the overall protective effect of alpha-thalassaemia status 
on risk of stroke in our patients remains to be determined 
and may differ from other populations less dominated by the 
Central African Republic (CAR) haplotype. No protective 



Table V. Relative risk ratios of 


genetic variants on category 


of vascular asymmetry as an indicator 


of vasospasm adjusted for a; 


;e and year of 


survey. 












Relative risk ratio (95% confidence interval)* 






Genotypes 


Mild vs. normal 


Moderate vs. normal 


Severe vs. normal 


N 


Aget 


0-08 (0-02-0-14) + 


-001 (-0-08-0-05) 


0 03 (-0 04-0 11) 


525 


Sext 


0-01 (-0-48-0-50) 


-0-08 (-0-58-0-42) 


0-04 (-0-54-0-62) 




Fetal Haemoglobinf 


-0 03 (-0-09-0-04) 


-0 02 (-0-09-0-04) 


-0 03 (-0 11-0 05) 




Haemoglobinf 


010 (-0-13-0-34) 


-0-27 (-0-50 to -0-03)+. 


-0-08 (-0-35-0-20) 




Haptoglobin, HP11 










HP12 


1-07 (0-58-1-98) 


1-12 (0-56-2-21) 


1-25 (0-56-2-79) 


410 


HP22 


0-86 (0-39-1-90) 


1-21 (0-53-2-75) 


1-12 (0-41-3-03) 




cx-Thalassaemia, normal 










1 Deletion 


1-69 (1-01-2-84) + 


0-98 (0-58-1-64) 


0-94 (0-52-1-70) 


549 


2 Deletions 


1-66 (0-86-3-23) 


0-99 (0-49-1-98) 


0-52 (0-20-1-35) 




G6PD Normal phenotype 










Mild (A-B) 


0-98 (0-48-1-98) 


0-94 (0-44-1-99) 


0-95 (0-39-2-31) 


564 


Affected (A-Z or A-A-) 


0-77 (0-38-1-58) 


1 11 (0-57-2-16) 


0-85 (0-35-2-03) 





*Degree of asymmerty is difference in the maximum velocities in LMCA and RMCA calculated as a percentage of the lower velocity. Mild 
75%, moderate -75-180%, severe >180%. 

f Results for multivariable regression including the marked variables. 
JP-value <0 05; Adjusted for survey year. 



40- 



© 2014 The Authors. British Journal of Haematology Published by John Wiley & Sons Ltd. 
British Journal of Haematoiogy, 2014, 165, 699-706 



703 



S. E. Cox ef al 



effect of altha-thalassaemia was apparent for degree of asym- 
metry between the ipsilateral and contralateral MCA. The 
effect of the alpha-thalassaemia deletion may be mediated at 
least in part via increased Hb. Co-inheritance of the 3-7 dele- 
tion is consistently and quantitatively associated with 
decreased mean cell volume (MCV), mean cell haemoglobin 
(MCH), mean cell haemoglobin concentration (MCHC) and 
haemolytic markers, including in this population, (Cox et al, 
2013) and with decreased WBC (Bernaudin et al, 2008). HbS 
concentration is the primary determinant of HbS polymeri- 
zation (Eaton & Hofrichter, 1987). Thus the protective effect 
of co-inheritance of the 3-7 deletion has been commonly 
assumed to be due to decreased sickling rates (Ballas, 2001) 
and consequent haemolysis. However, in the analysis by 
Bernaudin et al (2008) there were independent effects on 
CBFv of the 3-7 deletion after adjustment for MCV (MCH & 
MCHC not analysed), white blood cell count and the haemo- 
lytic marker lactate dehydrogenase (LDH) (itself indepen- 
dently associated in the multivariate model). The authors 
suggested increased red cell deformability as a potential 
mechanism. In a recent, large, carefully controlled study, co- 
inheritance of the 3-7 deletion in SCA was quantitatively and 
negatively associated with the proportion of dense dehy- 
drated red cells (%DRBCs) measured at steady state. 
Increased%DRBC was, in turn, positively associated with risk 
of priapism, leg ulcers and renal dysfunction, of which only 
renal dysfunction was associated with haemolysis (Bartolucci 
et al, 2012). There is evidence to suggest that the increased 
rigidity of DBRCs may promote vasoconstriction via reduced 
shear stress-induced ATP release as a vasodilatory signalling 
molecule (Wan et al, 2008). 

It is unlikely that the lack of an effect of the HP*2allele 
was due to a type II error as a result of a lack of power, as 
evidenced by our adequate sample size of 83 HP22 and 103 
HP 11 compared to our sample size estimate of 90 per 
group to detect an effect equivalent to half a standard devi- 
ation (SD) in CBFv. Thus although there was a probable 
increased variance in our data due to different observers 
between survey years, a similar effect size was observed for 
alpha-thalassaemia in this study (oc-/ot- (JV = 94) vs. ora/cta 
(N = 258), P-coefficient -16-14 cm/s = 0-4 SD of CBFv). 
However, the 95% CIs of the observed effect of HP22 did 
just include the effect size in the sample size calculation 
(—15-3 cm/s vs. 0-5 x SD = 20-9). Thus, it remains a possi- 
bility that small effects of the HP*2allele exist, reducing risk 
of either both or the lower extremes of CBFv, not captured 
in the multinomial logistic model, which was not included 
in pre-study sample size calculations. Thus despite the 
strong evidence for a mechanism whereby the HP22 variant 
could be expected to increase vascular activation and vaso- 
constriction in the inflammatory and haemolytic condition 
of SCA, our data does not support this suggestion. An 
explanation for this could be that HP is overwhelmed in 
SCA, such that phenotypic variations in its functions cease 
to be relevant. This scenario is supported by our observation 



that during well, steady-state clinic visits, HP levels were 
non-detectable in 96% of our patients (lower limit of 
detection = 0-04 g/1, N = 838) compared to 17% of non- 
SCA controls and not affected by genotype (S. E. Cox, J. 
Makani & A. M. Prentice, unpublished data). A possible 
alternative explanation is that in HP22 individuals the haem- 
oxygenase-1 compensatory pathway may be more successfully 
up-regulated in response to haem exposure, demonstrated to 
have powerful anti-inflammatory and anti-stasis effects (Jison 
et al, 2004; Belcher et al, 2006). 

Conflicting reports of an effect of G6PD on CBFv (Ber- 
naudin et al, 2008, 2011; Rees et al, 2009; Miller et al, 2011; 
Thangarajh et al, 2012) may result from variations between 
populations in either the phenotypic expression compared to 
assessed genotype or from population or methodological dif- 
ferences between in-vivo vs. in-vitro enzyme activity, when 
this has been measured directly (Johnson et al, 2009). There 
is little evidence for an association of either low enzyme 
activity (Miller et al, 2011) or genotype (Flanagan et al, 
2011) with stroke. Decreased enzyme activity was associated 
with TCD abnormality in children resident in France 
(Bernaudin et al, 2008) and this effect appeared to be inde- 
pendent of haemolysis. However, this association was not 
observed in England (Rees et al, 2009). Genotypes rsl050828 
or rsl050829 were associated with magnetic resonance angi- 
ography evidence of vasculopathy in a cohort from the US, 
England and France but children with TCD abnormality were 
excluded (Thangarajh et al, 2012). It is possible that G6PD 
polymorphisms play a role in the initiation or development 
of vasculopathy in certain environments but we have no 
evidence for this in our population resident in Africa and the 
lack of an association with stroke suggests that other risk 
factors may be more important. 

In conclusion, despite adequate power to determine a clin- 
ically relevant effect size of the _HP*2allele on CBFv, we could 
determine no evidence of such an effect. In a population of 
SCA patients resident in Africa, we confirmed the effect of 
alpha-thalassaemia, but could not find an effect of G6PD. 

Acknowledgements 

We warmly thank the patients and staff of MNH and 
MUHAS, Dar-es-Salaam, Tanzania who made this work 
possible. We also thank Gurishaeli Walter and Abdulnar 
Maupa for TCD measurements in 2009, Alex Macharia and 
Rachel Wiltshire for assistance with the genotyping, Jose- 
phine Mgaya and Harvest Mariki for haemoglobin quantifi- 
cation by HPLC, Lazarus Mramba and Anthony Fulford for 
statistical discussions and Tom Williams for his review of the 
manuscript. SEC, JM, FJK, CRJN 8c AMP designed the 
research. JM, DS, VSE, EK & AAB performed the research. 
AAB contributed essential analysis tools and data. SEC, FJK 
8c VSE analysed the data. SEC 8c FJK wrote the paper. All 
authors contributed to drafting and/or critical revisions of 
the manuscript and approved the final version. 



704 



© 2014 The Authors. British Journal of Haematology Published by John Wiley & Sons Ltd. 

British Journal of Haematology, 2014, 165, 699-706 



Polymorphisms & Transcranial Doppler in SCA 



Funding 

This work was supported by the Wellcome Trust UK: (SC) 
project grant 080025; (JM) personal fellowship 072064; stra- 
tegic awards 084538 and 095009. 



Supporting Information 

Additional Supporting Information may be found in the 
online version of this article: 

Table SI. Proportions of subjects for each combination of 
the three genotypes under investigation. 



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